The threat of terrorist action using biological warfare (BW), chemical or infectious agents has occurred throughout the world. These acts of terrorism are unpredictable and counter efforts have been aimed at rapid, accurate diagnosis and speedy treatment. Determination of the exact toxin that a subject has been exposed to is critical to treatment. More over, immediate determination of the exact toxin exposure is necessary to prevent irreparable damage, incapacity and death.
Current methods for pathogen or toxin identification require specialized reagents that are structural-based probes. For bio-engineered toxic agents, those probes may prove to be ineffective. The increased sophistication available for design of potential biological weapons will require reliance on better approaches to adequately identify such threats. Simple identification of toxins or infectious agents may be complicated by the fact that genetic manipulations could (1) make BW agents unrecognizable by structural-based technologies, or (2) enhance their devastating effects, making them toxic at undetectable levels. Furthermore, small amounts of common bacterial products, such as protein A or endotoxin, have been shown to markedly potentate activities of biological warfare threat toxins. The difficulties of identifying toxins experienced in the past could lead to potentially disastrous delays in responding appropriately to the threat or to the possibility of inappropriate treatment based on inadequate information. Thus far, diagnoses could only be made based on symptcoms, which may take 4-24 hours or more to appear, and by that time, the damage is irreversible and death may result.
Description of a Selected Group of Toxic Agents:
There are many toxic agents that are a threat to humans in situations of biological warfare. For example, SEB: Staphyloccocal enterotoxin B is a potent bacterial toxin known to cause lethal shock. The mode of exposure could be aerosol, food or water contamination. It interacts with the lymphoid cells, proximal tubule (PT) kidney and other cells initiating cascades of reactions ultimately leading to lethal shock. The initial symptoms for SEB-induced intoxication are vertigo, muscle weakness (vasoconstriction in the extremities) within 1-8 hrs of exposure to the toxin. The symptoms that follow are nausea, vomiting and diarrhea, along with hypotension and vasodilation of blood vessels in kidney and other organs (1-24 h). Respiratory distress and pathological hypotension eventually lead to irreversible shock and death at about 40-60 hrs post exposure, although very early incidents (ca. 6 h) have been observed. The mechanism of its action is not clear, nor is it understood how SEB is massively potententated by trace levels of contaminants such as Protein A or endotoxin. In short, there is no system available to determine host exposure or individual responses and the toxin is rapidly (30 min) removed from the blood stream to the kidney PT (75%), liver and spleen.
Anthrax is another highly toxic agent. Anthrax is a natural disease of herbivorous animals that can be transmitted to humans. The causative agent Bacillus anthracis, can form spores which are extremely hardy and can remain alive for a very long time. After inhalation of a heavy dose of anthrax spores, however, the onset of the disease may occur within a day and death may follow rapidly in a couple of days. The molecular changes caused by this agent in the host is totally unknown, therefore identifying genes altered by this agent is very crucial for rapid and effective detection.
Brucella is a highly infectious bacteria that causes disabling syrnptomatology (fever, chills, fatigue) in humans. Bacteria can be acquired through inhalation, ingestion, or penetration of damaged skin. As facultative intracellular parasites of macrophages, they primarily localize in the reticuloendothelial system. Bacteremia and symptoms occur from several days to several weeks after infection, presumably as a result of amplification of bacterial numbers in spleen, liver and bone marrow. Host response involves both Th1 and Th2 immune mechanisms, but is generally tilted toward Th1. In murine models of brucellosis, both antibody and T cells transfer immunity. Brucella LPS is relatively nonpyrogenic compared to LPS fiom Enterobacteriaceae. This property may explain the relative paucity of immune and inflammatory response early in infection
Plague is still another threatening toxic agent to man. The Y. pestis is an organism that causes plague. Plague symptoms include fever, chills, headache, hemoplysis and toxemia. This eventually leads to respiratory failure and death. Until now, diagnosis has been made by symptom analysis. This means that the progress of the illness can go unchecked before treatment is sought and is therefore, unsuccessful. A faster test is needed for plague.
Botulinum toxin is extremely potent neurotoxins produced by different strains of the bacterium Clostridium botulinum. There are seven serotypes of botulinum toxins, which share the same functional mechanism: they have an endopeptidase activity that cleaves a protein in synaptic vesicles thereby inhibiting release of acetycholine. The resulting block in neurotransmitter release causes general skeletal muscle paralysis with death occurring due to respiratory failure. Following inhalation or ingestion of botulinum toxin, symptoms may appear within 24 to 36 hours or may take several days to appear. This toxin causes weakness, dizziness, dry mouth and throat, blurred vision and diplopia, dysarthria, disphonia, dysphasia and respiratory failure. A faster test for exposure to the botulinum toxin is needed.
Cholera Toxin (CT) causes vomiting, headache, diarrhea resulting in death. Mortality is as high as 80%. Diagnosis is done by symptoms of diarrhea and dehydration. The Cholera Toxin is a very difficult toxin to spot in a blood sample. Therefore, a faster, non-symptom related test is needed to prevent death.
There is no easy or fast detection method to confirm the exposure to these and other toxic agents. The deadly symptoms of lethal shock appear before they are diagnosed so the important life-saving treatment is delayed which results in deaths that could be prevented if an earlier test were available. Current methods for pathogen identification using structural-based probes may not be useful for early diagnosis for the reasons stated above.
Therefore, an object of the present invention is to provide for a method of diagnosing exposure to toxic agents by measuring distinct patterns in the levels of expression of specific genes.
Another object is to provide a library of host gene expression responses to toxins such as anthrax, botulinum, Brucella, plague and cholera in comparison with altered gene expression in response to the staphylococcal enterotoxins and lipopolysaccharide (SEB).
It is a further object of the invention to select a panel of genes, the altered pattern of expression of which will provide a fingerprint that is indicative of exposure to a particular toxic agent, has the potential to reveal the severity of exposure and the individual susceptibility to the agent, and can provide indicators of course of impending illness for even unknown toxic agents.
A still further object of the invention is to utilize peripheral blood lymphoid cells from exposed individuals since these cells are a readily accessible reservoirs of historical information and they show unique patterns of responses although they may not be the primary target of a toxin.
A further object of the invention is to determine host functional responses to toxic agent exposure prior to onset of symptoms or illness.
A still further object of the invention is to provide a method of early treatment of subjects exposed to toxins with the intervention of drugs or with agents, such as antisense codes, which turn off the expression of genes that react detrimentally to toxins, based on the newly found gene changes.